NLRX1 Negatively Regulates TLR-Induced NF-ΚB Signaling by Targeting TRAF6 And

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NLRX1 Negatively Regulates TLR-Induced NF-kB Signaling by Targeting TRAF6 and IKK

Xiaojun Xia,1,5 Jun Cui,1,5 Helen Y. Wang,1 Liang Zhu,1 Satoko Matsueda,1 Qinfu Wang,1 Xiaoang Yang,1 Jun Hong,1 Zhou Songyang,3 Zhijian J. Chen,4 and Rong-Fu Wang1,2,* 1Center for Cell and Gene Therapy 2Departments of Pathology and Immunology Baylor College of Medicine, Houston, TX 77030, USA 3Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA 4Howard Hughes Medical Institute, Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA 5These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.immuni.2011.02.022

SUMMARY ligand-receptor binding generally leads to activation of common downstream pathways such as NF-kB, MAPK, and type I inter- Tight regulation of NF-kB signaling is essential for feron to induce cytokine and chemokine gene expression, which innate and adaptive immune responses, yet the facilitate pathogen clearance. Tight regulation of these key molecular mechanisms responsible for its negative signaling pathways is essential for both innate and adaptive regulation are not completely understood. Here, we immunity; otherwise, aberrant immune responses may occur, report that NLRX1, a NOD-like receptor family mem- leading to severe or even fatal bacterial sepsis, autoimmune, ber, negatively regulates Toll-like receptor-mediated and chronic inflammatory diseases (Karin et al., 2006; Liew et al., 2005). NF-kB activation. NLRX1 interacts with TRAF6 or IkB TLR activation usually results in the recruitment of adaptor kinase (IKK) in an activation signal-dependent fash- molecules, such as MyD88 and TRIF, which acts on a series ion. Upon LPS stimulation, NLRX1 is rapidly ubiquiti- of downstream signaling molecules such as TRAF6, which nated, disassociates from TRAF6, and then binds to possesses E3 ubiquitin ligase activity. Activated TRAF6 the IKK complex, resulting in inhibition of IKKa and synthesizes lysine 63 (K63)-linked polyubiquitin chains on itself IKKb phosphorylation and NF-kB activation. Knock- and other proteins, which then serves as a scaffold to recruit down of NLRX1 in various cell types markedly TAK1 and the IkB kinase (IKK) complex through binding to the enhances IKK phosphorylation and the production TAB2 and NEMO, respectively (Skaug et al., 2009). In contrast, of NF-kB-responsive cytokines after LPS stimula- activation of RIG-I and MDA-5 by double-stranded RNAs tion. We further provide in vivo evidence that or certain viruses results in recruitment of the MAVS protein NLRX1 knockdown in mice markedly enhances sus- (mitochondrial antiviral signaling; also called VISA, IPS-1, and Cardif) (Kawai et al., 2005; Meylan et al., 2005; Seth et al., ceptibility to LPS-induced septic shock and plasma 2005; Xu et al., 2005). These signaling events converge on IKK IL-6 level. Our study identifies a previously unrecog- complex to trigger activation of NF-kB signaling pathway and nized role for NLRX1 in the negative regulation of elicit inflammatory responses (Chen, 2005; Ha¨ cker and Karin, TLR-induced NF-kB activation by dynamically inter- 2006)(Akira et al., 2006)(Hayden and Ghosh, 2008). The IKK acting with TRAF6 and the IKK complex. complex consists of two catalytic subunits, IKKa and IKKb, and one regulatory subunit, NEMO (also known as IKK-g). Activation of the IKK complex involves phosphorylation of INTRODUCTION serine residues on the activation loop of IKKa/b, and binding of NEMO to polyubiquitin chains formed by upstream signals The innate immune system serves as the first line of defense (Skaug et al., 2009). However, the molecular mechanisms against invading pathogens, such as bacteria and viruses, by involved in the negative regulation of IKK activation are not recognizing a limited but highly conserved set of molecular well understood. structures, so-called pathogen-associated molecular patterns NLRX1, a member of the NLR family of proteins, contains an (PAMPs) (Akira et al., 2006). Recognition of such PAMPs relies N-terminal X domain, a nucleotide binding oligomerization do- on several classes of pattern recognition receptors (PRRs), main (NOD) and a C-terminal leucine-rich repeat domain (LRR). including Toll-like receptors (TLRs), NOD-like receptors (NLRs), NLRX1 was shown to be a negative regulator of RIG-I-mediated and RIG-I-like receptors (RLRs) (Akira et al., 2006; Meylan antiviral response, by binding to MAVS on mitochondria and et al., 2006; Rehwinkel and Reis e Sousa, 2010; Schroder and blocking its interaction with RIG-I (Moore et al., 2008). NLRX1 Tschopp, 2010; Takeuchi and Akira, 2010; Ting et al., 2010). also modulates TNF-a and Shigella infection-induced reactive Although different receptors recognize specific ligands, the oxygen species (ROS) release (Tattoli et al., 2008), but its

Immunity 34, 843–853, June 24, 2011 ª2011 Elsevier Inc. 843 Immunity NLRX1 Inhibits NF-kB Signaling by Targeting IKK

involvement in TLR-mediated NF-kB activation remains conditions, we determined IL-6 production in wild-type (WT) unknown. Here, we demonstrate that NLRX1 is a negative and MAVS-deficient mouse embryonic fibroblasts (MEFs) after regulator of TLR-mediated NF-kB signaling. In unstimulated LPS or poly(I:C) (a TLR3 ligand) treatment (Sun et al., 2006). As cells, NLRX1 interacts with TRAF6, but after LPS stimulation, shown in Figure 1C, IL-6 production was increased after LPS it undergoes K63-linked polyubiquitination and binds directly or poly(I:C) treatment, but such increases were potently inhibited to the IKK complex and inhibits the phosphorylation of IKK, by NLRX1 in both WT and MAVS-deficient MEFs, strongly thus inhibiting NF-kB activation and proinflammatory cytokine suggesting that NLRX1 inhibits IL-6 production through release. We further provide in vivo evidence showing that a MAVS-independent pathway. specific knockdown of NLRX1 in mice enhances LPS-indsuced Because the mouse NLRX1 protein shares 75% amino acid proinflammatory cytokine induction and renders mice more similarity with human NLRX1, we examined whether the inhibi- sensitive to LPS-induced septic shock. Thus, NLRX1 may serve tory function of NLRX1 is conserved between humans and as a useful target for manipulating immune responses against mice. Murine NLRX1 expression markedly inhibited NF-kB-luc infectious or inflammation-associated diseases, including activation by different stimuli, suggesting a conserved biological cancer. function by which NLRX1 regulates the NF-kB pathway (Fig- ure S1C). To test whether the findings in 293T cells can be RESULTS extended to other cells, we performed NF-kB-luc reporter assays in MEFs and THP-1 cells (human monocyte cell line), NLRX1 Negatively Regulates TLR-Induced NF-kB which express endogenous TLR4. LPS treatment induced strong Activation NF-kB-luc activity in both cell lines, and such activity was To identify new molecules that might negatively regulate potently inhibited when NLRX1 was cotransfected (Figure 1D). the NF-kB signaling pathway, we used a NF-kB-luciferase We further examined endogenous NF-kB DNA binding ability (NF-kB-luc) assay to screen a panel of candidates, including by a gel mobility shift assay. IKKb expression activated endoge- NLR family members, for their abilities to regulate NF-kB activity. nous NF-kB to bind to DNA probe containing NF-kB binding TLR4, the NF-kB-luc reporter, an internal control Renilla sites, but this activity was completely inhibited by NLRX1. By luciferase reporter (pRL-TK-luc), and the candidate genes were contrast, p65-mediated NF-kB DNA-binding activity was not cotransfected into HEK293T (293T) cells, which were then affected by NLRX1, which is consistent with the luciferase treated with the TLR4 ligand LPS for 18 hr for activation of reporter assay (Figure 1E). Since NF-kB activation is also asso- NF-kB-luc activity. One of the proteins identified that inhibited ciated with p65 translocation from the cytoplasm into the NF-kB activation was NLRX1, also known as NOD9 (Figure 1A). nucleus of cells after LPS stimulation, we examined p65 translo- We further confirmed NLRX1 inhibited NF-kB activation by cation in cells with or without NLRX1 expression. In cells trans- LPS at both early (6 hr) and late time points (24 hr) (Figure S1A fected with empty vector, p65 rapidly translocated from the available online). Similar inhibitory effects were observed with cytoplasm to the nucleus after LPS treatment. By contrast, p65 293T/TLR7 cells treated with CL-097 (a TLR7 ligand) when remained in the cytoplasm of the cells expressing NLRX1 after NLRX1 was coexpressed (Figure 1A). NLRX1 was recently LPS treatment (Figure 1F). Taken together, these results suggest shown to inhibit RIG-I-mediated type I interferon pathway by that NLRX1 inhibits TLR-induced NF-kB activation in various cell interacting with MAVS and sequestering it from interaction with types. RIG-I (Moore et al., 2008). Consistent with previous findings, we showed that NLRX1 inhibited RIG-I or MAVS-induced NLRX1 Dynamically Interacts with TRAF6 and the IKK IFN-b-luc activity (Figure S1B). Because most TLRs use Complex in Response to LPS MyD88 as the adaptor protein for downstream signal transduc- To determine the molecular mechanisms by which NLRX1 nega- tion (Takeuchi and Akira, 2010), we next tested whether NLRX1 tively regulates NF-kB signaling, we tested whether NLRX1 could inhibit MyD88-mediated NF-kB activation, which is directly interacts with the TRAF family adaptor proteins because independent of the MAVS-mediated pathway. As shown in different NF-kB activation signals use distinct TRAF proteins to Figure 1B, MyD88 strongly activated NF-kB-luc activity in 293T activate NF-kB signaling. Immunoprecipitation experiments cells, but such activation was potently inhibited by NLRX1, showed that NLRX1 interacted with TRAF6 and TRAF3, but not suggesting that it inhibits MyD88-mediated NF-kB activation. with TRAF2 or TRAF5 (Figure 2A). To determine whether NLRX1 also inhibits NF-kB activation To further determine the endogenous protein interactions under induced by downstream signaling molecules engaged in TLR physiological conditions, we developed an antibody against signaling, we cotransfected 293T cells with TRAF6, TAK1/ NLRX1 and confirmed its specificity in 293T cells, RAW264.7 TAB1, IKKa, IKKb, or NF-kB p65 subunit to activate the cells, and primary macrophages (Figure S2A–S2D). 293T/TLR4 NF-kB-luc reporter gene. We found that the activation of cells transfected with Flag-tagged NLRX1 (F-NLRX1) were treated NF-kB by TRAF6, TAK1, IKKa, and IKKb was markedly inhibited with LPS and the cell lysates were harvested at different time by NLRX1, but not by the NLR family members NOD1 or NOD2 points. Anti-TRAF6 immunoprecipitation followed by anti-Flag (Figure 1B). By contrast, NLRX1 did not inhibit p65-mediated immunoblotting revealed that NLRX1 interacted with TRAF6 in NF-kB activation, indicating that it may inhibit the NF-kB unstimulated cells, but dissociated from TRAF6 after LPS stimula- pathway immediately upstream of p65, most likely by interfering tion (Figure 2B). Similar results were obtained from RAW264.7 with the IKK complex. cells after LPS treatment (Figure 2C). To determine whether To test the possibility that MAVS is required for NLRX1- NLRX1 interacts with endogenous TRAF3, we performed similar mediated inhibition of NF-kB signaling under physiological experiments with anti-TRAF3, and found no endogenous

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Figure 1. NLRX1 Inhibits TLR-Induced NF-kB Activation (A) 293T cells were transfected with NF-kB-luciferase (luc) reporter plasmid and TLR4 or TLR7 plasmids, together with an pcDNA3.1 empty vector or NLRX1 construct, and analyzed for NF-kB-dependent luciferase activity (fold induction) after treatment with LPS (TLR4 ligand) or CL-097 (TLR7 ligand) for 18 hr. (B) 293T cells were transfected with NF-kB-luc, MyD88, TRAF6, TAK1+TAB1, IKKa, IKKb,orp65, along with NLRX1, NOD1,orNOD2 and analyzed for NF-kB-dependent luciferase activity at 36 hr posttransfection. (C) WT and MAVS/ MEFs were transfected with an empty vector or murine NLRX1 expression vector and then treated with LPS or poly (I:C). Cell supernatants were used for measuring IL-6 release by ELISA. (D) MEFs and human THP-1 cells were transfected with the NF-kB-luc reporter plasmid, along with an empty vector, murine NLRX1, or human NLRX1, and then analyzed for NF-kB-dependent luciferase activity after LPS treatment. (E) 293T cells were transfected with IKKb or p65 plasmids, together with an empty vector or NLRX1 plasmid. Nuclear extracts were used for detecting endogenous NF-kB DNA binding activity by a gel-mobility shift assay. OCT-1-DNA complexes served as a loading control. (F) RAW264.7 cells were transfected with an empty vector or murine NLRX1 plasmid. The localization of p65 was determined by immunostaining after 15 min of LPS treatment. Data from (A)–(D) are plotted as means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, versus controls. All experiments were performed at least three times.

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Figure 2. NLRX1 Dynamically Interacts with TRAF6 and IKK Complex upon LPS Treatment (A) 293T cells were transfected with F-TRAF2, F-TRAF3, F-TRAF5, F-TRAF6, and HA-NLRX1. Flag-tagged proteins were immunoprecipitated with anti-Flag followed by anti-HA immunoblotting. IP, IB, and WCL denote immunoprecipitation, immunoblotting, and whole-cell lysate, respectively. (B and C), Cell extracts of 293T/TLR4 cells transfected with Flag-NLRX1 (B) and RAW264.7 cells (C) were immunoprecipitated with anti-TRAF6, respectively, then analyzed together with WCL by IB with indicated antibodies. (D) 293T cells were transfected with F-IKKa, F-IKKb, F-NEMO, F-TRAF2, and HA-NLRX1. Flag-tagged proteins were immunoprecipitated with anti-Flag and blotted with anti-HA. (E–G) RAW264.7 cells (E), bone marrow-derived macrophages (BMMs; F), or peritoneal macrophages (G) were treated with LPS and the cell lysates were collected at the indicated time points and used for immunoprecipitation with anti-NEMO or anti-NLRX1, followed by immunoblotting with the indicated antibodies. Results are representative of three independent experiments.

NF-kB signaling by its disassociation from TRAF6 and interaction with TLR-activated IKK complex.

NLRX1 Is Ubiquitinated through a K63 Linkage after LPS Treatment Because NEMO binds to the K63-linked polyubiquitin chains of TRAF6 after LPS treatment, we reasoned that NLRX1 might undergo polyubiquitination, which might affect the dynamic interaction among NLRX1, TRAF6, and IKK. To test this possibility, we transfected 293T cells with plasmids expressing F-NLRX1 and interaction between NLRX1 and TRAF3 before or after LPS or HA-ubiquitin. Immunoprecipitation with anti-Flag and western poly(I:C) treatment, although the interaction between TRAF6 blotting with anti-HA revealed that NLRX1 was strongly ubiquiti- and NLRX1 was readily detected before LPS or poly(I:C) treatment nated (Figure 3A). Coexpression of NLRX1 with HA-ubiquitin (K48 (Figures S3A and S3B). These results suggest that NLRX1 inter- only) or HA-ubiquitin (K63 only) mutant revealed that NLRX1 was acts with TRAF6, but not TRAF3, in unstimulated cells. After preferentially ubiquitinated through K63-linked polyubiquitin activation, NLRX1 may switch from binding with TRAF6 to another chains (Figure 3A). Furthermore, we found that NLRX1 was target protein in the NF-kB signaling pathway to inhibit its activa- rapidly ubiquitinated through the K63 linkage by 10–15 min after tion. Given that NLRX1 inhibits NF-kB activation induced by LPS, LPS treatment in 293/TLR4 and MEF cells, but was then reduced. MyD88, TRAF6, TAK1, and IKK, but not by p65, we reasoned that By contrast, the K48-linked polyubiquitination of NLRX1 was not it might directly interact with the IKK complex. To this possibility, affected by LPS treatment (Figures 3B and 3C), raising the possi- we cotransfected 293T cells with HA-tagged NLRX1 together with bility that the polyubiquitin chains of NLRX1 is involved in the Flag-tagged IKKa, IKKb, and NEMO and tested their interaction. recruitment of NEMO and its IKK complex. Coimmunoprecipitation with anti-Flag and western blotting with To test this possibility, we ﬁrst determine the domains respon- anti-HA revealed that NLRX1 interacted with IKKa, IKKb, and sible for the interaction between NLRX1 and NEMO. We gener- NEMO, but not TRAF2 (Figure 2D). When IKK complex was immu- ated several deletion mutants of NLRX1 containing NOD and noprecipitated from RAW264.7 cell extracts with anti-NEMO, the LRR domains (NLRX1-NOD-LRR), LRR domain only (NLRX1- IKK-associated NLRX1 was barely detectable before LPS treat- LRR), the X and NOD domains (NLRX1-X-NOD), and the NOD ment, but the interaction was increased and then peaked at domain only (NLRX1-NOD) (Figure 3D). Immunoprecipitation 60 min after LPS treatment. Importantly, anti-NEMO immunopre- and western blot analyses revealed that NLRX1-FL, NLRX1- cipitation also pulled down IKKa and IKKb (Figure 2E), suggesting NOD-LRR, NLRX1-LRR, and NLRX1-X-NOD, but not NLRX1- that NLRX1 interacts with the IKK complex only after LPS stimu- NOD, could interact with NEMO (Figure 3E), suggesting that lation. Similar results were obtained when THP1 cells were treated the X and LRR domains are important for interaction with with LPS (Figure S3C). We also observed LPS-induced interaction NEMO. Interestingly, the mutants capable of interacting with between IKK and NLRX1 in bone marrow-derived macrophages, NEMO exhibited smear bands on the top of the gel, suggesting primary MEFs, and primary mouse peritoneal macrophages when that NEMO also binds to polyubiquitinated NLRX1. anti-NLRX1 was used for coimmunoprecipitation (Figures 2F NEMO contains an ubiquitin-binding domain (UBD), which and 2G; Figure S3D). These results suggest that NLRX1 inhibits facilitates its binding to polyubiquitinatin chains of adaptor

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showed that smeared bands of NRLX1 protein were observed in the immunoprecipitation with anti-NEMO in the cells transfected with NEMO FL, NEMO 44-419, and NEMO 86-419, which contain UBD domain. By contrast, NLRX1 pulled down with anti- NEMO in the cells transfected with NEMO mutants without UBD domain (i.e., NEMO 1-196 and NEMO 1-302) failed to show the smear bands (Figure 3F). These results suggest that the K63- linked polyubiquitination of NLRX1 is involved in the binding of NEMO to NLRX1, thus facilitating the initial recruitment of the NEMO/IKK complex to form a stable complex after LPS stimulation.

LRR Domain of NLRX1 Binds to the Kinase Domain of Activated, but Not Inactive, IKKb To determine how NLRX1 interacts with IKKa and IKKb, we transfected 293T cells with IKKa or IKKb with various NLRX1 deletion mutants and then performed coimmunoprecipitation experiments. Like NLRX1-FL, NLRX1-NOD-LRR, NLRX1-X-NOD, and NLRX1-NOD interacted with IKKa and IKKb, whereas NLRX1- LRR failed to interact with IKKa or IKKb (Figures 4A and 4B), suggesting that the NOD domain of NLRX1 is required for the binding of NLRX1 to IKKa and IKKb. However, functional assays with NF-kB-luc reporter showed that NLRX1 constructs containing LRR domain (i.e., NLRX1-NOD-LRR and NLRX1-LRR) had strong inhibitory activity, whereas NLRX1 mutants lacking LRR domain (i.e., NLRX1-X-NOD and NLRX1-NOD) exhibited weak or little inhibitory effect on NF-kB-luc activity (Figure 4C). These results suggest that the NOD domain of NLRX1 is involved in its interaction with IKK, but not for its inhibitory effect on IKKb- induced NF-kB-luc activity. By contrast, the LRR domain of NLRX1 could not bind to IKKa and IKKb, but is critically required for its inhibitory effect on IKKb-induced NF-kB-luc activity. Thus, a key question is how the NLRX1 LRR domain inhibits IKKb- induced NF-kB-luc activity, given that it could not bind to IKKb. One clue from the early results showing that NLRX1 binds to IKK only after LPS stimulation (Figures 2E–2G) raises the possi- Figure 3. NLRX1 Ubiquitination and Its Involvement in the Interac- bility that the NLRX1 LRR domain might bind to activated IKKa/ tion between NEMO and NLRX1 IKKb, rather than the inactive form of IKK. To test this possibility, (A) 293T cells were transfected with HA-ubiquitin, HA-ubiquitin (K48 only), we transfected 293T cells with NLRX1 mutants and IKKb EE HA-ubiquitin (K63 only), and F-NLRX1. Flag-NLRX1 was immunoprecipitated mutant, a constitutive active form containing serine to glutamic with anti-Flag beads and blotted with anti-HA. acid substitution on activation loop mimicking phosphorylation. (B) 293T/TLR4 cells transfected with F-NLRX1 were collected at the indicated time points after LPS treatment. Flag-NLRX1 was immunoprecipitated with anti- Indeed, we found that NLRX1-LRR could strongly bind to IKKb Flag beads and then analyzed by immunoblotting with indicated antibodies. EE (Figure 4D), suggesting that the NLRX1 LRR domain can (C) MEF lysates were collected at the indicated time points after LPS treat- bind to the activated IKKb. To further determine the mechanisms ment. Endogenous NLRX1 was immunoprecipitated with anti-NLRX1 and then by which the NLRX1 LRR domain binds the activated IKKb (IKKb analyzed by immunoblotting with anti-K63-linked polyubiquitin antibody. EE), we reasoned that the activated IKKb may undergoes confor- (D) Schematic diagram showing deletion constructs of NLRX1 containing mational changes and allow the LRR-interacting domain to be different domains. (E) 293T cells were cotransfected with Flag-NEMO and the indicated exposed, as previously proposed (Hayden and Ghosh, 2008). Myc-NRLX1 deletion constructs. Flag-tagged NEMO was immunoprecipitated To test this possibility, we generated several IKKb deletion with anti-Flag beads, and blotted with anti-Myc. mutants containing a kinase domain (KD), the leucine-zipper (F) 293T cells were cotransfected with HA-NLRX1 and the indicated Flag- domain (LZ), or helix-loop-helix domain (HLH). Coimmunopreci- NEMO deletion constructs. Flag-tagged proteins were immunoprecipitated pitation and western blot analyses revealed that NLRX1 inter- with anti-Flag beads and blotted with anti-HA. Results are representative of acted with the kinase domain of IKKb (IKKb-KD), but not with three independent experiments. IKKb-HLH or IKKb-LZ domain (Figure 4E). Importantly, we found that the LRR domain, but not other domains, of NLRX1 exhibited proteins and formation of protein complexes upon activation (Ea the strongest interaction with the kinase domain of IKKb et al., 2006). To test whether the UBD domain is required for (IKKb-KD) (Figure 4F), suggesting that the LRR domain directly NEMO binding to NLRX1, we performed experiments with binds to the kinase domain of IKKb when it becomes accessible NEMO deletion mutants. Coimmunoprecipitation experiments because of conformation changes after activation.

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Figure 5. NLRX1 Inhibits IKK Phosphorylation (A) 293T cells transfected with HA-IKKa, HA-IKKb, and HA-p38 with or without F-NLRX1 were used for analyzing the phosphorylation of IKKa/b and p38. (B) 293T/TLR4 cells transfected with increasing amount of F-NLRX1 plasmid were used to analyze the phosphorylation of IKKa/b after LPS treatment. (C) RAW264.7 cells were treated with LPS, and cell lysates were collected at the indicated time points for immunoprecipitation with anti-NLRX1 or anti- NEMO, then immunoblotted with indicated antibodies or kinase assay (KA). (D) 293T cells were transfected with HA-IKKb, together with an empty vector, F-NLRX1, or its deletion constructs, and the phosphorylation of IKK was determined. Results are representative of three independent experiments.

NLRX1 Inhibits IKKa and IKKb Phosphorylation and NF-kB Activation To determine the mechanisms by which NLRX1 inhibits NF-kB activation, we next cotransfected 293T cells with IKKa or IKKb together with an empty vector or a NLRX1 expression vector. As shown in Figure 5A, expression of IKKa or IKKb protein strongly induced IKK autophosphorylation, as detected by the phospho-specific IKK antibody. However, expression of NLRX1 markedly inhibited the phosphorylation of IKKa and IKKb. By contrast, the phosphorylation of p38 was not affected by NLRX1 expression (Figure 5A), indicating that NLRX1 specifically inhibits IKK phosphorylation, but not p38 phosphorylation. Furthermore, NLRX1 inhibits LPS-induced IKK phosphorylation Figure 4. Mapping the Interaction Domains of NLRX1 and IKK in 293T/TLR4 cells in a dose-dependent manner (Figure 5B). Subunits Because NLRX1 or its LRR domain preferentially binds to a b (A and B) 293T cells were transfected with HA-IKK (A) or HA-IKK (B) and the activated form of IKKb and inhibits IKKb-induced NF-kB-luc indicated F-NLRX1 deletion constructs. HA-tagged IKKa or HA-IKKb was immunoprecipitated with anti-HA beads and blotted with anti-Flag. activity (Figures 4C–4F), we next sought to determine the (C) 293T cells were transfected with NF-kB-luc, IKKb, along with F-NLRX1 and phosphorylation and kinase activity of IKKa/b in association its deletion constructs and analyzed for NF-kB-dependent luciferase activity. with either NEMO or NLRX1 in LPS-stimulated RAW264.7 cells. Data are plotted as means ± SD. We first immunoprecipitated NEMO-associated IKK complex (D) Experiment performed as in (B), except that IKKb WT was replaced by IKKb with anti-NEMO and NLRX1-associated IKK complex with EE mutant form. (E) Identification of the kinase domain of IKKb interacting with NLRX1. The upper panel shows a schematic diagram showing protein domain structures of proteins were immunoprecipitated with anti-Flag beads and then blotted with the IKKb deletions. KD, kinase domain; LZ, leucine zipper; HLH, helix-loop- anti-HA. helix. As shown in the lower panel, 293T cells were cotransfected with (F) Experiment performed as in (B), except IKKb WT form was replaced by IKKb HA-NLRX1 with F-IKKb and its deletion constructs are indicated. Flag-tagged KD mutant. Results are representative of three independent experiments.

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Figure 6. Knockdown of NLRX1 Enhances IKK Phosphor- ylation and NF-kB-Responsive Cytokine Gene Expression (A) RAW264.7 cells were transfected with control siRNA or NLRX1-specific siRNA, and then treated with LPS for the indicated time points. LPS-induced IKK, IRF3, and MAPK (p38, JNK, and ERK) activation were measured by IB with phosphospecific antibodies and IKK activity was measured by KA. (B) Quantitative comparison of signaling activation between NLRX1 knockdown and control cells by density scanning of the blots in (A). (C) RAW264.7 cells were transfected with control siRNA or NLRX1-specific siRNA, and then treated with LPS. The cytokine Tnf-a and Il-6 gene expression in RAW264.7 cells induced by LPS at different time points were determined by real-time PCR. (D) Production of cytokine TNF-a and IL-6 in culture medium of RAW264.7 cells transfected NLRX1-specific or control siRNAs after LPS treatment. Data in (C and D) are plotted as means ± SD; *p < 0.05, **p < 0.01, versus controls. All experiments were performed at least three times with similar results.

RAW264.7 cells transfected with control siRNA. Kinase activity assays also showed increased and prolonged IKK kinase activity for GST-IkBa phosphorylation in NLRX1 knockdown cells compared to control siRNA-treated cells (Figure 6A). By contrast, MAPK (p38, JNK, and ERK) signaling activation was largely unchanged in the cells transfected with NLRX1-specific or control siRNAs (Figure 6A). Simi- larly, LPS treatment induced IRF3 activation, but there was no difference in IRF3 phosphorylation anti-NLRX1, respectively, and then determined their IKK phos- between NLRX1 siRNA and control siRNA-treated cells (Fig- phorylation and kinase activities for IkBa phosphorylation. We ure 6A). To quantify these results, we did density scanning of found that that NEMO-associated IKK complex displayed IKK the protein bands, and found that only IKK phosphorylation phosphorylation and kinase activity for IkBa phosphorylation, and kinase activity exhibited significant differences between as expected. By contrast, anti-NLRX1 immunoprecipitates NLRX1 knockdown and control cells. There were no appre- contain IKK (IKKa/IKKb and /NEMO) complex after LPS treat- ciable differences in JNK, p38, EKR, and IRF3 phosphorylation ment, but did not exhibit any IKK phosphorylation or kinase between NLRX1 knockdown and control cells (Figure 6B). activity for IkBa phosphorylation (Figure 5C). Consistent with Conversely, LPS-induced IKK phosphorylation was markedly the results in Figure 4C, we found that the LRR domain-contain- inhibited when NLRX1 was overexpressed in RAW264.7 cells ing NLRX1 constructs (i.e., NLRX1-FL, NLRX1-NOD-LRR, and (Figure S4B). Together, these results suggested that specific NLRX1-LRR) strongly inhibited IKKb phosphorylation, whereas knockdown of NLRX1 enhances LPS-induced IKK phosphory- NLRX1-X-NOD and NLRX1-NOD exhibited weak or no inhibitory lation and NF-kB activation. activity (Figure 5D). Taken together, these results suggest that To determine whether increased NF-kB activation in the once NLRX1 (specifically its LRR domain) binds to the exposed NLRX1 knockdown cells could increase NF-kB-responsive kinase domain of activated/phosphorylated IKK, NLRX1-associ- cytokine genes, we performed knockdown experiments in ated IKK complexes lose the phosphorylation probably through RAW264.7 cells and found that Tnf-a and Il-6 gene expression unknown phosphatases as well as their kinase activity for IkBa were significantly higher in NLRX1 knockdown cells than those phosphorylation under physiological conditions. in control siRNA-transfected cells after LPS treatment (Fig- ure 6C). Consistent with these results, the cytokine level of Knockdown of NLRX1 Enhances IKK Phosphorylation TNF-a and IL-6 was increased in NLRX1 knockdown cells and NF-kB-Responsive Genes compared to control cells (Figure 6D). Furthermore, NF-kB- Because NLRX1 inhibits IKK phosphorylation and NF-kB acti- responsive Ccl2 and Cxcl10 gene expression was also enhanced vation, we reasoned that knockdown of NLRX1 would increase in NLRX1 knockdown cells compared to control cells (Fig- IKK phosphorylation and NF-kB-responsive gene expression. ure S4C). Similar results were observed with THP-1 cells trans- To test this prediction, we knocked down NLRX1 expression fected with NLRX1-specific and control siRNAs (Figures S4D by NLRX1-specific siRNA in RAW264.7 cells and stimulated and S4E). Taken together, these results suggest that specific the cells with LPS (Figure S4A) and found that specific knock- knockdown of NLRX1 increases IKK phosphorylation and down of NRLX1 markedly enhanced IKK phosphorylation, NF-kB-responsive cytokine gene expression in response to especially at 60 min after LPS stimulation, compared with LPS treatment.

Immunity 34, 843–853, June 24, 2011 ª2011 Elsevier Inc. 849 Immunity NLRX1 Inhibits NF-kB Signaling by Targeting IKK

Figure 7. NLRX1 Negatively Regulates NF-kB Signaling and Cytokine Response In Vivo (A) Knockdown of murine NLRX1 expression in different tissues of NLRX1-KD mice as measured by quantitative real-time PCR. (B) NLRX1 protein expression was determined by immunoblotting in WT and NLRX1-KD MEFs. WT and NLRX1-KD MEFs were infected by VSV-eGFP and then visualized by ﬂuorescent microscopy. (C) WT and NLRX1-KD mice (n = 10 per group) were injected intravenously with poly(I:C) (200 mg per mouse) and then sera were collected at indicated times for IFN-b measurement. (D) LPS-induced IKK and MAPK activation in peritoneal macrophages from WT and NLRX1-KD mice. The quantiﬁed results are shown after band density scanning.

(E) IL-6 production in WT and NLRX1-KD MEFs after treatment with LPS, Pam3CSK4 or infected with VSV-eGFP. (F) IL-6 and TNF-a production in peritoneal macrophages from NLRX1-KD or WT mice after treatment with LPS. (G) Survival of NLRX1-KD and WT mice (n = 9 per group) after peritoneal injection with LPS (30 mg/kg). (H) Plasma IL-6 levels from WT and NLRX1-KD mice (n = 9 per group) at 3 hr after peritoneal injection with LPS (30 mg/kg). Data in (A, C, E, and F) are plotted as means ± SD. *p < 0.05, **p < 0.01, versus controls. All experiments were performed at least three times with similar results.

NLRX1 Negatively Regulates NF-kB Signaling over, in MEFs generated from NLRX1-KD mice, we found a and Cytokine Response In Vivo marked reduction of NLRX1 protein expression compared to To substantiate the physiological function of NLRX1 in vivo, we WT MEFs (Figure 7B). It has been reported that knockdown of generated NLRX1 knockdown (NLRX1-KD) transgenic mice human NLRX1 in 293T cells enhances antiviral immunity and constitutively expressing shRNA that speciﬁcally knocked inhibits virus replication (Moore et al., 2008). To determine down mouse NLRX1 (Figure S5A). By comparison with the ubiq- whether knockdown of mouse NLRX1 expression also contrib- uitous expression of NLRX1 in the tissues of wild-type (WT) mice, utes to antiviral immune responses, we infected MEFs with ves- mRNA transcript levels were markedly reduced in the thymus, ticular stomatitis virus (VSV)-enhanced green ﬂuorescence spleen, and bone marrow of NLRX1-KD mice (Figure 7A). More- protein (eGFP). VSV-eGFP infection was strongly inhibited in

850 Immunity 34, 843–853, June 24, 2011 ª2011 Elsevier Inc. Immunity NLRX1 Inhibits NF-kB Signaling by Targeting IKK

NLRX1-KD MEFs compared with WT cells, as evaluated by GFP plasma IL-6 level (Figure 7H). However, we did not observe signal in the cells (Figure 7B). Similar results were obtained in significant difference in plasma TNF-a level (data not shown). Nlrx1/ MEFs (Figure S5B). NLRX1-KD mice also produced These results provide in vivo evidence that NLRX1 negatively more interferon-b (IFN-b) in plasma, compared with WT mice regulates NF-kB activation, NF-kB-responsive cytokine IL-6, after intravenous injection of poly (I:C) (Figure 7C). To further and LPS-induced septic shock. determine whether NLRX1 knockdown affects mouse survival in response to viral infection, we injected NLRX1-KD and WT DISCUSSION mice with VSV-eGFP via the tail vein and then monitored their survival. NLRX1-KD mice showed slightly but not significantly NLRX1 is a member of the NOD-like family of proteins, which are increased mouse survival compared to WT mice (Figure S5C). characterized by a conserved NOD and LRR regions, and are Consistent with this observation, we did not detect appreciable involved in the activation of diverse signaling pathways (Akira differences in serum viral titers and cytokine (IFN-b and IL-6) et al., 2006; Inohara et al., 2005; Meylan et al., 2006; Ting and levels (Figures S5D–S5F). Together, these results suggest that Davis, 2005). Among these proteins, NOD1, NOD2, and NALP3 NLRX1 knockdown has some effects on type I interferon and have been extensively studied and shown to activate inflamma- antiviral immunity in vitro, but not in vivo, thus raising the possi- some pathway once they encounter relevant PAMPs (Akira et al., bility that NLRX1 may have a predominant regulatory role in TLR- 2006; Inohara et al., 2005; Kobayashi et al., 2005; Meylan et al., induced NF-kB signaling. 2006; Shaw et al., 2008; Ting and Davis, 2005). However, the bio- To test this possibility, we isolated peritoneal macrophages logical function of other NLR proteins remains poorly understood from WT and NLRX1-KD mice and examined IKK phosphoryla- (Ting et al., 2010). Previous report shows that NLRX1 functions tion and MAPK signaling activation after LPS treatment. Con- as a negative regulator of cellular antiviral immunity by interfering sistent with the results obtained with RAW264.7 cells, IKK with viral infection-induced RIG-I-MAVS complex formation or phosphorylation was much higher and prolonged in NLRX1-KD modulates Shigella infection-induced reactive oxygen species macrophages compared to WT cells after LPS treatment, (ROS) generation (Moore et al., 2008; Tattoli et al., 2008). Our whereas p38 and JNK phosphorylation was largely unchanged studies, however, indicate that NLRX1 functions as a negative (Figure 7D). To define the physiological function of NLRX1 in regulator that inhibits TLR-induced NF-kB activation. The find- response to other TLR ligands, we found that IL-6 production ings presented here identify a previously unrecognized role for was enhanced in NLRX1-KD MEFs after stimulation with LPS, NLRX1 in the negative regulation of NF-kB signaling by targeting

Pam3CSK4 (a TLR2 ligand), and infection with VSV-eGFP, TRAF6 and IKK complex. Furthermore, we provide compelling compared to WT MEFs (Figure 7E). Similarly, we found that evidence that NLRX1 negatively regulates NF-kB signaling, cyto- IL-6 expression was increased in Nlrx1/ MEFs compared to kine production, and LPS-induced septic shock in vivo.

WT MEFs treated with LPS, Pam3CSK4, poly (I:C), or CL-097 In response to TLR stimulation, most TLRs recruit MyD88 and (Figures S6A and S6B). Furthermore, IL-6 and TNF-a production activate the downstream molecules such as TRAF6, which acts in NLRX1-KD macrophages was significantly higher than those as an E3 ubiquitin ligase to catalyze K63-linked polyubiquitin in WT cells after treatment with LPS, Pam3CSK4, poly (I:C), chain on itself. The polyubiquitin chains of TRAF6 function as a CL-097, and CpG (a TLR9 ligand) (Figure 7F; Figures S6C and scaffold to recruit the TAK1 and IKK complexes through binding S6D). These results suggest that NLRX1-KD and Nlrx1/ cells to the regulatory subunits TAB2 and NEMO, respectively (Skaug markedly enhance NF-kB signaling and cytokine (IL-6 and et al., 2009). TLR-induced NF-kB signaling pathway has been TNF-a) production after TLR stimulation. well characterized, but it is still not completely understood that To determine the specificity of NLRX1 in inhibiting NF-kB acti- how the activated signals are dampened to prevent pathological vation, we compared NF-kB activation in NLRX1-KD and WT consequences, such as septic shock and inflammatory disor- cells induced by TNFR, T cell receptor (TCR), or B cell receptor ders. The increased list of negative regulators of TLR signaling (BCR) stimulation. We did not observe an appreciable difference (Komuro et al., 2008; Liew et al., 2005) includes diverse proteins in IL-6 production between NLRX1-KD and WT cells after TNF-a that regulate the TLR signaling pathway at different stages. The treatment (Figure S6E). This result is consistent with immunopre- deubiquitinating enzymes A20 and CYLD inhibit NF-kB signaling cipitation experiments showing that NLRX1 interacted neither by targeting TRAF6 upstream of IKK (Kovalenko et al., 2003; with TRAF2 nor with IKK in cells treated with TNF-a (Figure 2A Liew et al., 2005; Trompouki et al., 2003; Wertz et al., 2004), while and Figure S6F). Furthermore, we failed to find differences in a recent report shows that CUEDC2 inhibits IKK activity by re- TCR- or BCR-induced NF-kB signaling between NLRX1-KD cruiting a phosphatase PP1 and keeps IKK in an inactivated and WT cells (Figures S6G and S6H), suggesting that NLRX1 is status (Li et al., 2008). We recently reported that NLRC5 inhibits not involved in TCR- or BCR-induced NF-kB activation. TLR-induced NF-kB activation by constitutive interaction with We next sought to determine the function of NLRX1 in LPS- IKKa/b, but not NEMO (Cui et al., 2010). However, our results induced septic shock. NLRX1-KD and WT mice were injected here show that NLRX1 interacts with TRAF6 in unstimulated with a high dose of Escherichia coli LPS (30 mg/kg) intraperitone- cells, but disassociates from TRAF6 upon LPS stimulation. By ally and monitored for their survival. As shown in Figure 7G, more contrast, there was no interaction between NLRX1 and TRAF2 than 80% of the NLRX1-KD transgenic mice died within 18 hr, or TRAF3 before or after stimulation. In addition, NLRX1 did whereas all WT mice remained alive. All NLRX1-KD transgenic not show any inhibitory effect on TNFR-, TCR-, or BCR-induced mice died within 30 hr compared to 50% of the WT mice, which NF-kB signaling, which is independent of TRAF6. These results continued to survive after 36 hr (Figure 7G). Enhanced LPS further suggest that NLRX1 specifically inhibits TLR-induced toxicity in NLRX1-KD mice correlated with markedly increased TRAF6-dependent NF-kB signaling through targeting TRAF6.

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Although the molecular mechanisms responsible for its disasso- ical function of NLRX1 in vivo, we generated NLRX1-KD mice with ciation from TRAF6 are not known, we found that NLRX1 is shRNA targeting NLRX1 expression. Our study using NLRX1-KD rapidly ubiquitinated at 10–15 min after LPS stimulation. Interest- mice, as well as cells derived from both NLRX1-KD and NLRX1 ingly, like TRAF6, NLRX1 also undergoes K63-linked, but not knockout mice, further provide evidence that NLRX1 is a critical K48-linked, polyubiquitination after LPS stimulation. The interac- negative regulator of TLR-induced NF-kB signaling pathway. tion between NLRX1 and the IKK complex is induced after LPS Macrophages and MEFs from NLRX1-KD mice enhance IKK stimulation. Thus, the molecular mechanisms by which NLRX1 phosphorylation and produce more proinflammatory cytokines inhibits NF-kB signaling are distinct from NLRC5. It appears that in response to TLR stimulation. More importantly, NLRX1-KD several negative regulators specifically target TRAF6 and/or mice are more sensitive to LPS-induced septic shock, with asso- IKK to control NF-kB activation through distinct mechanisms. ciation of increased level of plasma IL-6. Although we observed Our results show that the K63-linked polyubiquitination of enhanced antiviral immunity against VSV-eGFP infection in NLRX1 may be involved in the dynamic interaction of NLRX1 NLRX1-KD and Nlrx1/ MEFs and increased IFN-b production with TRAF6 or with the IKK complex. NLRX1 polyubiquitination in NLRX1-KD mice after poly(I:C) injection, we did not observe may alter its interaction with TRAF6 and serve as a scaffold to appreciable differences in mouse survival, viral titers, and serum recruit the IKK complexes through binding to the regulatory cytokine levels after VSV-eGFP infection. These results suggest subunit NEMO. This notion is further supported by our results that NLRX1 may play a predominant role in TLR-induced NF-kB showing that NEMO mutants lacking UBD domain fail to pull signaling, compared with type I IFN pathway. down the polyubiquitinated NLRX1 (the smear bands). Further- Collectively, the findings presented here identify a previously more, NLRX1 interacts only with constitutively active IKKb. IKK unrecognized role for NRLX1 in the negative regulation of activation has been proposed to depend on phosphorylation of NF-kB signaling and provide insights into molecular mecha- IKKb on its activation loop, which in turn might induce the com- nisms by which NLRX1 dynamically interacts with TRAF6 and plex conformation change and exposure of kinase domain (Hay- IKK in a signal-dependent fashion. Thus, NLRX1 may serve as den and Ghosh, 2008). Indeed, we show that the LRR domain in a therapeutic target for the treatment of infectious and autoim- NLRX1 interacts strongly with the kinase domain of IKKb when mune diseases, as well as cancer. IKKb becomes accessible as a result of conformational changes after IKK activation or phosphorylation. These results suggest EXPERIMENTAL PROCEDURES that the interactions of NLRX1 with TRAF6 or with the IKK complex are complicated and are signaling dependent. On the Transfection and Reporter Assay 293T cells, MEFs, and THP-1 cells were transfected with plasmids encoding basis of these findings, we propose a working model: NLRX1 NF-kB luciferase or IFN-b luciferase, pRL-TK Renilla luciferase, and different associates and disassociates with TRAF6, depending on the expression or control vectors. Lipofectamine 2000, lipofectamine LTX with activation status of cells. Upon stimulation with LPS, both PLUS reagent (Invitrogen, Carlsbad, CA), and Amaxa nucleofector kit V (Lonza TRAF6 and NLRX1 are rapidly ubiquitinated through the K63 Amaxa, Gaithersburg, MD) were used for tranfection of 293T cells, MEFs, and linkage and they dissociate from each other. The K63 linked THP-1 cells, respectively. The luciferase activity was determined by a dual polyubiquitination of TRAF6 leads to the recruitment of the luciferase assay kit (Promega, Madison, WI) with a Luminoskan Ascent TAK1 complex and IKK complex to polyubiquitin chains, acti- luminometer (Thermo Scientific, Waltham, MA). vating IKK for its phosphorylation, which becomes the target of Immunoprecipitation and Immunoblot Analyses NLRX1. The polyubiquitin chains of NLRX1 may serve as a scaf- For immunoprecipitation experiments, whole-cell extracts were prepared after fold to recruit NEMO, and then NLRX1 binds to activated IKK and transfection or stimulation and incubated with indicated antibodies together forms a relatively stable NLRX1-IKK complex through the inter- with Protein A/G beads (Pierce, Rockford, IL) for overnight. For anti-Flag action between the NLRX1 LRR domain and the IKKb kinase or anti-HA immunoprecipitation, anti-Flag or anti-HA agarose gels (Sigma, domain. Meanwhile, the phosphorylation of IKK is removed by St. Louis, MO) were used. Beads were then washed four times with lysis buffer, unknown phosphatases, resulting in formation of a NLRX1-IKK and immunoprecipitates were eluted with SDS loading buffer (Cell Signaling Technology, Danvers, MA) and resolved in SDS-PAGE gels. The proteins complex without IKK phosphorylation and kinase activity. were transferred to PVDF membrane (Bio-Rad) and further incubated with Thus, the interactions of NLRX1 with TRAF6 or with the IKK com- the indicated antibodies. LumiGlo Chemiluminescent Substrate System from plex are a dynamic process and rely on TLR-induced activating KPL (Gaithersburg, MD) was used for protein detection. signal. To determine whether TRAF6 is responsible for NLRX1 ubiquitination, we performed experiments with WT and Traf6/ Generation of NLRX1-KD Transgenic Mice MEFs and found that NLRX1 is equally ubiquitinated in both Plasmid DNA for NLRX1-specifc shRNA1 from Open biosystems (Huntsville, types of cells after LPS treatment (data not shown), suggesting AL) was linearized by restriction enzymes PvuI and XbaI digestion and injected into fertilized mouse eggs of C57BL/6 strain through a Transgenic Core. that TRAF6 may not be the E3 ligase responsible for NLRX1 ubiq- Positive founders and offspring were identified by genotyping PCR of the tail uitination. Thus, further studies are needed to identify such E3 DNA with the primers 50-ACGTCGAGGTGCCCGAAGGA-30 (forward) and ubiquitin ligases for the ubiquitinatination of NLRX1. 50-AAGCAGCGTATCCACATAGCGT-30 (reverse). Three transgenic lines were Although NLRX1 has been reported as a mitochondria-associ- maintained by crossing founders to C57BL/6. All the mice were maintained ated protein, it is evident that some NLRX1 protein is present in in a pathogen-free animal facility. All animal studies performed were approved the cytoplasm to function as a negative regulator of NF-kB sig- by the BCM Institutional Animal Care and Use Committee. naling. Similar proteins such as Stat3 and Bcl-2 have been Statistical Analysis reported to have different biological functions in the cytoplasm Unless indicated otherwise, all data were plotted as means ± SD. Significant and mitochondria (Gough et al., 2009; Hockenbery et al., 1993; differences between groups were determined by two-tailed Student’s t test Jacobson et al., 1993; Wegrzyn et al., 2009). To define the biolog- or two-way ANOVA. In the mouse endotoxic shock study, Kaplan-Meier

852 Immunity 34, 843–853, June 24, 2011 ª2011 Elsevier Inc. Immunity NLRX1 Inhibits NF-kB Signaling by Targeting IKK

survival curves were generated and analyzed for statistical signiﬁcance with Komuro, A., Bamming, D., and Horvath, C.M. (2008). Negative regulation of Graphpad Prism 4.0 software. cytoplasmic RNA-mediated antiviral signaling. Cytokine 43, 350–358. Kovalenko, A., Chable-Bessia, C., Cantarella, G., Israe¨ l, A., Wallach, D., and SUPPLEMENTAL INFORMATION Courtois, G. (2003). The tumour suppressor CYLD negatively regulates NF-kappaB signalling by deubiquitination. Nature 424, 801–805. Supplemental Information includes Supplemental Experimental Procedures Li, H.Y., Liu, H., Wang, C.H., Zhang, J.Y., Man, J.H., Gao, Y.F., Zhang, P.J., Li, and six ﬁgures and can be found with this article online at doi:10.1016/ W.H., Zhao, J., Pan, X., et al. (2008). Deactivation of the kinase IKK by CUEDC2 j.immuni.2011.02.022. through recruitment of the phosphatase PP1. Nat. Immunol. 9, 533–541. Liew, F.Y., Xu, D., Brint, E.K., and O’Neill, L.A. (2005). Negative regulation ACKNOWLEDGMENTS of toll-like receptor-mediated immune responses. Nat. Rev. Immunol. 5, 446–458. We thank J. Ting for Nlrx1 / MEF cells, M. Karin, B. Su, S.-C. Sun, and M. Kunihiro for IKKa, IKKb, NEMO, p38, p100, and TAB1 constructs, and Meylan, E., Curran, J., Hofmann, K., Moradpour, D., Binder, M., H. Shu for TRAF constructs. We would also like to thank A. Aboseda Alagbala Bartenschlager, R., and Tschopp, J. (2005). Cardif is an adaptor protein in for generating bone marrow-derived macrophages and critical reading of the the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437, manuscript. This work was supported in part by grants from National Cancer 1167–1172. Institute, National Institutes of Health and Cancer Research Institute. Meylan, E., Tschopp, J., and Karin, M. (2006). Intracellular pattern recognition receptors in the host response. Nature 442, 39–44. Received: November 14, 2010 Moore, C.B., Bergstralh, D.T., Duncan, J.A., Lei, Y., Morrison, T.E., Revised: January 31, 2011 Zimmermann, A.G., Accavitti-Loper, M.A., Madden, V.J., Sun, L., Ye, Z., Accepted: February 19, 2011 et al. (2008). NLRX1 is a regulator of mitochondrial antiviral immunity. Nature Published online: June 23, 2011 451, 573–577. Rehwinkel, J., and Reis e Sousa, C. (2010). RIGorous detection: Exposing virus REFERENCES through RNA sensing. Science 327, 284–286.

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NLRX1 Negatively Regulates TLR-Induced NF-&Kappa;B Signaling by Targeting TRAF6 And

NLRX1 Negatively Regulates TLR-Induced NF-ΚB Signaling by Targeting TRAF6 And